SEPTIN2 suppresses an IFN-γ-independent, proinflammatory macrophage activation pathway

Interferon-gamma (IFN-γ) signaling is necessary for the proinflammatory activation of macrophages but IFN-γ-independent pathways, for which the initiating stimuli and downstream mechanisms are lesser known, also contribute. Here we identify, by high-content screening, SEPTIN2 (SEPT2) as a negative regulation of IFN-γ-independent macrophage autoactivation. Mechanistically, endoplasmic reticulum (ER) stress induces the expression of SEPT2, which balances the competition between acetylation and ubiquitination of heat shock protein 5 at position Lysine 327, thereby alleviating ER stress and constraining M1-like polarization and proinflammatory cytokine release. Disruption of this negative feedback regulation leads to the accumulation of unfolded proteins, resulting in accelerated M1-like polarization, excessive inflammation and tissue damage. Our study thus uncovers an IFN-γ-independent macrophage proinflammatory autoactivation pathway and suggests that SEPT2 may play a role in the prevention or resolution of inflammation during infection.

a. GFP intensity data obtained from high-content screening were analyzed to investigate regulatory factors in the IFN-γ-independent hyperpolarization.Seventeen genes (14 increased in the VSV infection group and 3 increased in the HSV-1 infection group) are shown.n = 3 in each group (a).b-d.The body weight (b), numbers of macrophages isolated from spleen (c) and lung (d) of Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl mice (without infection).n = 9 in each group (b-d).e. Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl mice were infected with 1×10 7 PFU VSV or 1×10 8 PFU HSV-1.Daily injection of αIFN-γ (12 mg/kg) or its isotype control from 1 day before infection.The secretion of IFN-γ in BALF at 7 dpi was detected.n = 6 in each group (e).LOD: limit of detection.f.Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl mice were infected with 1×10 7 PFU VSV.Viral burdens in lungs at 1, 4 and 7 dpi were detected.g.PMs were infected with VSV (MOI = 1) for 6 and 12 hours.The viral titres were measured by plaque assays.n = 6 in each group (f, g).h.Gating strategy for analysis of innate immune cell populations.i,j. qRT-PCR analysis of upregulation markers ( Il6, Il12b, Cxcl9 and Ptgs2) (i) and downregulation markers (Mrc1, S100a4, Ube2c and Slc9a9) (j) of M1like polarization in Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl PMs after being infected with VSV (MOI = 1) for 6 hours.The data were normalized to GAPDH expression (i, j).n = 6 in each group (i, j).
Data are shown as the mean ± s.e.m. (a-g, i, j).One-way ANOVA followed by Bonferroni post hoc test (a-g, i, j) was used for data analysis.Abbreviation: UI, uninfected.Source data are provided as a Source Data file.CHX (50 µg/mL) was used to inhibit the protein synthesis.The expression of HSPA5 was detected by western blotting.g.Immunoblot analysis of HSPA5 in PMs after being pretreated with E64d (10 μg/mL) + PepA (10 μg/mL) or MG132 (10 μg/mL) for Quantitative data are graphed in (j).k.The acetylation of HSPA5 modified by SEPT2 was identified using an in vitro acetylation system described in Fig. 4f.n = 3 in each group (h-k).l.Screening of potential acetylase by siRNAs in iBMDMs after being infected with VSV (MOI = 1) for 12 hours.The acetylation of HSPA5 was analyzed.m.Validation of Atat1 +/+ and Atat1 -/-iBMDMs.n, o.Immunoprecipitation analysis of acetylated HSPA5 in iBMDMs after being infected with HSV-1 (MOI = 5) (n).
Data are shown as the mean ± s.e.m. (c, d, j, o-q).One-way ANOVA followed by  (a, c-e, i-k, n, o) and the median ± interquartile (m).One-way ANOVA followed by Bonferroni post hoc test ( a, c-e, i-k
Antibodies were diluted in in 1% BSA (pan Phospho-Serine/Threonine antibody) or 5% skim milk (others).For coimmunoprecipitation (except ubiquitin immunoprecipitation), cell lysates were obtained according to the standard cell lysis method.Lysates were centrifuged and the supernatants were incubated with appropriate antibodies and Protein A-Agarose (Santa Cruz Biotechnology) overnight at 4°C.Precipitated protein complex was mixed with 5× SDS Loading Buffer and boiled at 98°C for 3 min, followed by immunoblotting with indicated antibodies.The uncropped blots are provided in the Source Data file.

Tandem ubiquitin binding entity analysis
Tandem ubiquitin binding entities (TUBEs, UM401M, LifeSensors, Malvern, PA, USA) were used for ubiquitin immunoprecipitation.Cells were harvested and lysed in specific cell lysis buffer [50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 1 mM EDTA, 1% NP-40 and 10% glycerol].Then, equilibrated magnetic-TUBEs were added into cell lysate (100 µL resin/1 mg protein) and incubated for 2 hours at 4º C on a rocker platform.After washing for three times with TBST, beads were collected using a Detection of the accumulation of unfolded proteins NTPAN-MI probe was resynthesized as previously described 5 and used to analyze the accumulation of unfolded proteins.The resynthesis process was performed strictly according to the reference.The pure product was obtained after silica-gel chromatography in 62% yield as a yellow solid and identified by 1

ROS production detection
H2DCFDA (D399, Invitrogen) was used for oxidative stress measurement according to the manufacturer's instruction.Briefly, PMs obtained from Sept2 fl/fl and Sept2 fl/fl Lyz2-Cre mice were infected with VSV (MOI = 1) for the indicated times.The 1400W•dihydrochloride (100 μM) was used to inhibit iNOS activity.H2DCFDA (10 µM ) was added into the cell culture medium and incubated at 37°C for 20 min.The reactive oxygen species production was determined by detecting the fluorescence in D for anti-HSPA5 antibody and pan Acetyl-Lysine antibody) in the Probemaker PLA Probe Diluent and add the primary antibody solutions to each slide.The slides were incubated at 4°C for 8 hours, followed by ligation, amplification and detection.After final washing, the acetylation and ubiquitination levels of HSPA5 were identified by fluorescence intensity.

Protein complex structure analysis
The structures of ATAT1 and SCNN1B were first predicted by AlphaFold2, and the inactive sites were removed.Since HSPA5 has template information with high similarity, the prediction model of HSPA5 was obtained by homology modeling with SwissDock.Next, ZDOCK and HADDOCK were used to achieve protein-protein rigid docking, and RosettaDock was used to achieve protein-protein flexible docking.
The preliminary conformation of the global docking was optimized with Rosetta in two rounds.The results were quality checked according to the Rosetta evaluation metric.After screening by the scoring function, biochemical experimental data and empirical knowledge, the final models of ATAT1/HSPA5 and SCNN1B/HSPA5 complexes were obtained.The binding interfaces of the two complexes were comprehensively characterized and systematically analyzed using interaction analysis platforms PDBsum and PLIP.

Isothermal titration calorimetry
ITC was used to identify the affinity of ATAT1 or SCNN1B to HSPA5 as previously described 6 .Purified proteins were transferred to buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, and 2 mM β-mercaptoethanol by HiTrap desalting column (GE healthcare, Atlanta, GA, USA).Titrations were performed by using a Malvern Microcal PEAQ-ITC calorimeter at room temperature.Data were analyzed using the PEAQ-ITC analysis software.

Surface plasmon resonance
SPR was used to identify the affinity of ATAT1 or SCNN1B to HSPA5 as previously described 7 .Purified HSPA5 protein (20 μg/mL, in 10 mM sodium acetate buffer, pH 5.0) was coupled on a CM5 Chip (GE Healthcare) using amine coupling method.
Different doses of ATAT1 or SCNN1B proteins (0 to 2,000 nM) were injected for association and dissociation analysis.The dissociation rate constants were calculated using steady state affinity obtained for each enzyme concentration.

Fluorescent resonance energy transfer
FRET system was built and performed as shown in Figure S6J-K following a previous report with partial modification 8 .Briefly, CMV-HSPA5(TAG)-ATAT1-YFP (or CMV-HSPA5(TAG)-SCNN1B-YFP) was transfected into Atat1 -/-Scnn1b -/-iBMDMs, followed by transfection of different doses of SCNN1B (or ATAT1).A fluorescent amino acid ANAP was incorporated into Phenylalanine 452 of HSPA5.The fluorescence of ANAP-HSPA5 was detected in the 410-530 nm range with excitation at 405 nm, and YFP-ATAT1 (or YFP-SCNN1B) fluorescence was monitored in the 550-620 nm range with excitation at both 405 and 488 nm.FRET signals arising from binding of HSPA5 to ATAT1 (or SCNN1B) were monitored in the 550-620 nm range with excitation of ANAP at 405 nm.FRET ratio (IYFP/IANAP) was recorded from single live cell image at the indicated times.

Dual-luciferase reporter assay
HEK-293FT cells were seeded on 24-well plate and cultured overnight to reach 75% confluency.Luciferase reporter plasmids (300 ng) and internal control plasmid pRL-SV40 (10 ng) were cotransfected into cells for 24 hours.Afterwards, cells were lysed for dual-luciferase detection.The relative luciferase activity was measured by firefly luciferase luminescence divided by renilla luciferase luminescence.All the primers used for luciferase reporter plasmids construction are listed in Supplementary Data 3.

Chromatin immunoprecipitation assay
ChIP assay was performed using SimpleChIP Plus Enzymatic Chromatin IP Kit (9004S, Cell Signaling Technology).Cells were fixed with 1% formaldehyde to crosslink histone and non-histone proteins to DNA, and then pellet nuclei was collected by centrifugation at 2,000×g for 5 min at 4°C.Next, micrococcal nuclease (0.5 µL per 4 ×10 6 cells) was added to the pellet nuclei.The lysate was incubated at 37°C for 20 min with frequent mixing to digest chromatin to a length of approximately 150-900 bp.Then lysate was subjected to sonication to break nuclear membrane.Incubate samples for 30 s on wet ice between pulses.Afterwards, clarify lysate by

a.
Survival of Sept2 fl/fl Lyz2-Cre (n = 15) and Sept2 fl/fl (n = 11) mice intraperitoneally infected with 1×10 8 PFU HSV-1.Daily injection of αIFN-γ (12 mg/kg) from 1 day before infection to the end of the experiments was performed to block IFN-γ signaling.b-e.Flow cytometry analysis of iNOS, CD80 and CD86 in Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl PMs after being infected with RNA viruses (SeV [MOI = 1], EMCV [MOI = 1]) (b), or DNA viruses (HSV-1 [MOI = 5], Adv [MOI = 1]) (c) for 12 hours, respectively.Quantitative data are graphed in (c, e).n = 3 in each group (b-e).f, g.Flow cytometry analysis of Arg-1, CD163 and CD206 in Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl PMs after being infected with VSV (MOI = 1) or HSV-1 (MOI = 5) for 12 hours (f).The gating of Arg-1 high , CD163 high and CD206 high populations was determined against those of the uninfected control.Quantitative data are graphed in (g).n = 3 in each group (f, g).h-m.The knockdown efficiency of RNA interference was detected by western blotting.n-s.Quantitative flow cytometry data of iNOS, CD80 and CD86 in Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl PMs after transfection with the indicated siRNAs 12 hours after VSV (MOI =1, n, p, s), HSV-1 (MOI = 5, o, q) or EMCV (MOI = 1, r) infection.n = 3 in each group (h-s).Data are shown as Kaplan-Meier curves (a) or the mean ± s.e.m. (c, e, g, n-s).Logrank (Mantel-Cox) test (a) or one-way ANOVA followed by Bonferroni post hoc test (c, e, g, n-s) were used for data analysis.Abbreviation: NC, negative control.Source data are provided as a Source Data file.infections a, b.Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl mice were infected with 1×10 7 PFU VSV or 1×10 8 PFU HSV-1.The secretion of IFN-α, IFN-β in BALF at 7 dpi (a) and the mRNA expression of Mx1 and Isg15 in lung tissues at 7 dpi (b) were detected.The data were normalized to GAPDH expression (b).n = 6 in each group (a, b).LOD: limit of detection.c, d.The knockdown efficiency of IFNAR1 and IFNAR2 siRNAs was examined by western blotting.e, f.Quantitative flow cytometry data of iNOS, CD80 and CD86 in Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl PMs after transfection with the indicated siRNAs 12 hours after VSV infection (e, f).n = 3 in each group (c-f).g, h.Flow cytometry analysis of iNOS, CD80 and CD86 in Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl PMs after being infected with E.coil (MOI = 20), Listeria (MOI = 20), M.tuberculosis (MOI = 10) or stimulated with LPS (100 ng/mL) for 12 hours (g).The gating of iNOS high , CD80 high and CD86 high populations was determined against those of the uninfected control.Quantitative data are graphed in (h).n = 3 in each group (g, h).i. Cell viability of Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl PMs after being infected with VSV (MOI = 1) for the indicated times.n = 6 in each group (i).j, k.PMs were infected with VSV (MOI = 1) for 12 hours.The expression of full-length GSDMD and its cleaved form was detected by western blotting (j).n = 3 in each group (j).The apoptosis was detected by flow cytometry (k).n = 6 in each group (k).Data are shown as the mean ± s.e.m. (a, b, e, f, h, i, k).One-way ANOVA followed by Bonferroni post hoc test (a, b, e, f, h, i, k) was used for data analysis.Abbreviation: NC, negative control.Source data are provided as a Source Data file.knockdown in pLKO-Tet-on-SEPT2 shRNA stably transfected iBMDMs after VSV infection (MOI = 1).f-h.Relative expression of SEPT2 (f), iNOS activity (g) and proinflammatory cytokines levels (Tnfα, Il1b, Il6 and Il12b) (h) were detected at indicated time points.The qRT-PCR data were normalized to GAPDH expression (f, h).n = 3 in each group (f-h).Data are shown as the mean ± s.e.m. (b-d, f-h).One-way ANOVA followed by Bonferroni post hoc test (b-d, f-h) was used for data analysis.Abbreviation: UI, uninfected.NC, negative control.Source data are provided as a Source Data file.sequencing.GO analysis was used to determine the enrichment of pathways.c.Transmission electron microscopy of Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl PMs after being infected with VSV (MOI = 1) or HSV-1 (MOI = 5) for 12 hours.Green arrow: normal ER.Red arrow and circle: swollen ER and degranulated ribosomes.Scale bar = 5 μm.n = 3 in each group (c).d, e. Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl PMs were infected with VSV (MOI = 1).The XBP1 mRNA splicing levels were detected by qRT-PCR (d), and activation of M1-like polarization related signaling pathways were detected by immunoblots (e) at 6 and 12 hours post infection.n = 6 (d) or n = 3 (e) in each group.f.The chemical structural of NTPAN-MI probe.g.Sept2 fl/fl and Sept2 fl/fl Lyz2-Cre PMs were infected with VSV (MOI = 1) in the absence or presence of 4-PBA (5 mM) for 12 hours.The activation of PERK, IRE1 and ATF6 pathways was detected by western blotting.n = 3 in each group (g).h.The knockdown efficiency of PERK, IRE1 and ATF6 siRNAs.i. Sept2 fl/fl and Sept2 fl/fl Lyz2-Cre PMs were transfected with PERK, IRE1 or ATF6 siRNA for 24 hours, followed by infection of VSV (MOI = 1) for 12 hours.The activation of eIF2α pathway was detected by western blotting.n = 3 in each group (h, i).Data are shown as the mean ± s.e.m. (d).One-way ANOVA followed by Bonferroni post hoc test (d) was used for data analysis.Abbreviation: UI, uninfected.NC, negative control.Source data are provided as a Source Data file.50 μm.n = 3 in each group (a).b-f.Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl PMs were infected with VSV (MOI = 1) for 12 hours.b.The intracellular ROS levels were detected by H2DCFDA probe.c-f.1400W (100 μM) was used to treat cells during VSV infection.The inhibitory effect of 1400W on iNOS activity was shown in (c).The intracellular ROS (d), UPR levels (e) and proinflammatory cytokines (f) under 1400W treatment were detected by H2DCFDA probe, western blotting and ELISA, respectively.n = 6 (b-d, f) or n = 3 (e) in each group.g, h.Sept2 fl/fl Lyz2-Cre and Sept2 fl/fl PMs were infected with VSV for 12 hours.g.Intracellular lipid content was detected by BODIPY 647 staining.Scale bar = 50 μm.h.The expression of lipid metabolismrelated genes (Fas, Acc and Srebp-1c) was detected by qRT-PCR.The data were normalized to GAPDH expression (h).n = 3 (g) or n = 6 (h) in each group.Data are shown as the mean ± s.e.m. (b-d, f, h).One-way ANOVA followed by Bonferroni post hoc test (b-d, f, h) was used for data analysis.Abbreviation: UI, uninfected.Source data are provided as a Source Data file.by SCNN1B a.The knockdown efficiency of SEPT2 siRNAs in Sept2 fl/fl PMs.b, c. qRT-PCR (b) and immunoblot analysis (c) of HSPA5 in Sept2 fl/fl PMs after being transfected with SEPT2 siRNAs and infected with VSV (MOI = 1) for 12 hours.d, e. Immunoblot analysis (d) and qRT-PCR (e) of HSPA5 in Sept2 fl/fl Lyz2-Cre PMs after being transfected with HA-tagged SEPT2 and infected with VSV (MOI = 1) for 12 hours.n = 3 in each group (a-e).f.PMs obtained from Sept2 fl/fl Lyz2-Cre mice were transfected with Myc-tagged WT HSPA5 plasmids and infected with VSV (MOI = 1).

4
Fig. 3h was detected by western blotting.l.Schematic of WT and mutated ubiquitins Bonferroni post hoc test (c, d, j, o-q) was used for data analysis.Abbreviation: NC, negative control.Source data are provided as a Source Data file.a.Immunoprecipitation analysis of the interaction of ATAT1 and SEPT2 in iBMDMs after being infected with VSV (MOI = 1) for 12 hours.b, c.Immunoprecipitation analysis of SEPT2 oligomerization in iBMDMs after being transfected with HAtagged SEPT2 or Myc-tagged SEPT2.n = 3 in each group (a-c).d, e.Immunoprecipitation analysis of the interaction between SEPT2-HSPA5 complex and other SEPTINs in PMs after being infected with VSV (MOI = 1) for 12 hours.n = 3 in each group (d, e).f.The knockdown efficiency of SEPT6, SEPT7 and SEPT9.g.Quantitative flow cytometry data of iNOS, CD80 and CD86 in iBMDMs after being transfected with the indicated siRNAs for 24 hours and infected with VSV (MOI = 1) for 12 hours.h.iBMDMs were infected with VSV (MOI =1) for 12 hours.The colocalization of SEPT2 and SEPT7 was analyzed.Scale bar = 5 μm.i. iBMDMs were transfected with SEPT6, SEPT7 or SEPT9 siRNA for 24 hours, followed by infection of VSV (MOI = 1) for 12 hours.The expression of SEPT2 was detected.n = 3 in each group (f-i).j, k. iBMDMs were infected with VSV (MOI = 1) for 12 hours.The mRNA and protein expressions of SEPTINs were detected.l. iBMDMs were transfected with SEPT2, SEPT6 or SEPT9 siRNA for 24 hours, followed by VSV infection (MOI = 1) for 12 hours.The mitochondrial fission rates were detected by live cell imaging.n = 6 (j) or n = 3 (k) or n = 9 (l) in each group.m. iBMDMs were infected with VSV (MOI =1) for 12 hours.The co-localization of SEPT2 with HSPA5 and SEC61B was analyzed.Scale bar = 5 μm.n = 3 in each group (m).Data are shown as the mean ± s.e.m. (g, j, l).One-way ANOVA followed by Bonferroni post hoc test (g, j, l) was used for data analysis.Abbreviation: UI, a, b.The acetylation and ubiquitination levels of HSPA5 in iBMDMs after being transfected with Myc-tagged HSPA5, HSPA5 K327A, or SEPT2 siRNA and infected with VSV (MOI = 1) for 12 hours (a).Quantitative data are graphed in (b).n = 3 in each group (a, b).c.Quantitative data of the acetylation and ubiquitination levels of HSPA5 in iBMDMs after being stimulated with IFN-γ (50 ng/mL) and infected with VSV (MOI = 1) for 12 hours.n = 3 in each group (c).d.Quantitative data of the acetylation and ubiquitination levels of HSPA5 in NIH-3T3, L929 and TC-1 cells after being transfected with SEPT2 siRNA and infected with VSV (MOI = 1) for 12 hours.n = 3 in each group (d).e. f.The acetylation and ubiquitination levels of HSPA5 in PMs after being transfected with Myc-tagged HSPA5, HSPA5 K327A, or ATAT1 siRNA and infected with VSV (MOI = 1) for 12 hours (e).Quantitative data are graphed in (f).g, h.The acetylation and ubiquitination levels of HSPA5 in PMs after being transfected with Myc-tagged HSPA5, HSPA5 K327A, or SCNN1B siRNA and infected with VSV (MOI = 1) for 12 hours (g).Quantitative data are graphed in (h).i. PLA of the acetylation and ubiquitination status of HSPA5 in iBMDMs after being transfected with Myc-tagged HSPA5 or HSPA5 K327A and infected with VSV (MOI = 1).Scale bar = 20 μm.n = 3 in each group (e-i).j-l.Overall structure of the inactive structure-removed HSPA5 (j), ATAT1 (k) and SCNN1B (l).m, n.The rigid dock and flexible dock structures of ATAT1-HSPA5 (m) and SCNN1B-HSPA5 (n) complexes.o, p. Schematic of FRET analysis.MG132 (10 μg/mL) was used to inhibit the proteasomal degradation of HSPA5 (a-i).Data are shown as the mean ± s.e.m. (b-d, f, h).One-way ANOVA followed by Bonferroni post hoc test (c, d) was used for data analysis.Abbreviation: NC, negative control.Source data are provided as a Source Data file.a. H&E staining of lung lesions in C57BL/6J mice intraperitoneally infected with 1×10 4 PFU PR8M or PR8F at 7 dpi.Daily intraperitoneal injection of αIFN-γ (12 mg/kg) was performed to block IFN-γ signaling.The pathology scores were quantitated and shown as histogram.Scale bar = 400 μm.n = 6 in each group (a).b, c.Flow cytometry analysis of iNOS, CD80 and CD86 in C57BL/6J mice PMs after being infected with PR8M or PR8F (MOI = 1) for 12 hours (b).Quantitative data are graphed in (c).n = 3 in each group (b, c).d.The secretion of proinflammatory cytokines was detected by ELISA.n = 6 in each group (d).e. C57BL/6J mice PMs were infected with PR8M or PR8F (MOI = 1) for 12 hours.The expression of SEPT2 was detected.n = 6 in each group (e).f.Immunoblot analysis of SEPT2 and HSPA5 in iBMDMs after being infected with VSV (MOI = 1), HSV-1 (MOI = 5), or PR8M (MOI = 1) in the absence or presence of 4-PBA (5 mM) for 12 hours.g.Immunoblot analysis of SEPT2 in iBMDMs after being transfected with siRNAs targeting PERK, IRE1 and ATF6 and infected with VSV (MOI = 1), HSV-1 (MOI = 5), or PR8M (MOI = 1) for 12 hours.n = 3 in each group (f, g).h.Validation of Xbp1 -/-iBMDMs.n = 3 in each group (h).i. Dual-luciferase reporter assay of sXBP1-binding activity to SEPT2 promoter truncations in HEK-293FT cells.j.Dual-luciferase reporter assay of sXBP1-binding activity to WT and mutated 2.5kb SEPT2 promoter in HEK-293FT cells after being cotransfected with different doses of sXBP1 overexpression plasmids.n = 6 in each group (i, j).k.Chromatin immunoprecipitation assay of sXBP1-binding activity to SEPT2 promoter in iBMDMs after being infected with VSV (MOI = 1) for 12 hours.n = 6 in each group (k).l.Electrophoretic mobility shift assay of sXBP1-binding activity to SEPT2 probe.n = 3 in each group (l).m. qRT-PCR analysis of SEPT2 in PBMCs obtained from healthy individuals (n = 21, 10 males and 11 females), influenza patients without cytokine storm (n = 29, 19 males and 10 females) and influenza patients with cytokine storm (n = 25, 17 males and 8 females).n, o.PBMCs obtained from influenza patients with cytokine storm were treated with IXA4 (10 μM) or APY29 (1 μM) for 6 hours.The XBP1 mRNA splicing level (n) and the expression of SEPT2 (o) were detected.n = 12 in each group (n, o).Data are shown as the mean ± s.e.m.
) and Mann-Whitney U test (m-o) were used for data analysis.Abbreviation: UI, uninfected.EV, empty vector.Source data are provided as a Source Data file.Twenty-four hours later, cells were infected with VSV (MOI = 1) or HSV-1 (MOI = 5) for 12 hours.For screening of E3 ubiquitin ligase regulating HSPA5, siRNA (25 nM) targenting each gene and HSPA5::GFP vector were transfected into Sept2 fl/fl Lyz2-Cre PMs, followed by VSV infection (MOI = 1) for 12 hours.After that, cells were fixed with 4% paraformaldehyde (PFA) and stained with DAPI.Images were taken under the 20×and 40×objectives and two detection channels (FITC and DAPI) of the ImageXpress Pico system (Molecular Devices, Shanghai, China).Cells in each well were taken one image under 20×objective and three images under 40×objective for quantitative analysis.The mean GFP fluorescence intensity was calculated based on cell area and GFP fluorescence intensity.Statistical analysis was performed by CellReporterXpress automated imaging analysis software (Molecular Devices).The catalog of two siRNA libraries is listed in Supplementary Data 1 and Supplementary Data 2.
H NMR and 13 C NMR.For cell staining, NTPAN-MI was dissolved in DMSO as 2 mM stocks and diluted to 50 µM before use.Cells were treated with 50 µM NTPAN-MI for 30 min at room temperature and subsequently washed.DAPI was used to stain nuclei.Before imaging, cells were fixed with 4% PFA for 20 min.Images were acquired on a Leica DMi8 inverted fluorescence microscope.The fluorescence of NTPAN-MI was detected in the 527 nm range with excitation at 405 nm.DAPI fluorescence was monitored in the 460 nm range with excitation at 350 nm.The mean fluorescence intensity was quantitated by image J software.